This invention pertains to a translational control element placed in a vector to cause a selective translation of a toxin, including a toxin that acts by metabolizing a drug to become toxic (a xe2x80x9cconditional toxin,xe2x80x9d e.g., the herpes simplex virus type-1 thymidine kinase (HTK)/ganciclovir interaction), inside solid tumor cells, while leaving normal cells unaffected due to their inability to translate the toxin encoded by the vector.
The major determinant of morbidity and mortality for patients with a primary malignant tumor is the emergence and progression of metastatic islets resistant to conventional therapy. It has been estimated that at least 50% of patients presenting with a primary tumor already bear metastases at the time of diagnosis. See R. H. Goldfarb et al., xe2x80x9cTherapeutic agents for treatment of established metastases and inhibitors of metastatic spread: preclinical and clinical progress,xe2x80x9d Current Opinion in Oncology, vol. 4, pp. 1130-41(1992). Cancer gene therapy has developed as a means of attacking cancers resistant to conventional approaches. Much of the work has been directed at targeting characteristics of the primary tumor, with attention to choice of vector and transcriptional regulation. Two examples of this are the use of tissue-specific promoters and inducible promoters. See K. Binley et al., xe2x80x9cAn adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer,xe2x80x9d Gene Therapy, vol. 6, pp. 1721-1727 (1999). Despite some advances, these approaches have not successfully addressed the major problem of how to target metastases. Not only are metastases more difficult to reach but, due to their heterogeneity, they frequently do not maintain the specific gene expression pattern of the primary tumor, upon which gene therapy is generally designed. See S. J. Hall et al., xe2x80x9cCooperative therapeutic effects of androgen ablation and adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy in experimental prostate cancer,xe2x80x9d Cancer Gene Therapy, vol. 6, pp. 54-63 (1999). There is an unfilled need for an effective in vivo cancer gene therapy that permits selective killing of both the primary tumor and distant metastases, while distinguishing cancer cells from normal cells.
One of the main obstacles to gene therapy has been the difficulty of successfully targeting cancer cells, while not harming normal cells. Indeed, it has been found that even when therapeutic vectors are delivered locally to a primary tumor, systemic effects still often occur, indicating that the vector has become blood-borne. See Z. Long et al., xe2x80x9cMolecular evaluation of biopsy and autopsy specimens from patients receiving in vivo retroviral gene therapy, Human Gene Therapy, vol. 10, pp.:733-40 (1999); and M. Kaloss et al., xe2x80x9cDistribution of retroviral vectors and vector producer cells using two routes of administration in rats,xe2x80x9d Gene Therapy, vol. 6, pp. 1389-1396 (1999). One approach to circumvent this problem is to use elements allowing specific transcriptional regulation of the vector, e.g., the use of tissue-specific promoters and inducible promoters. See Binley et al., 1999; and L. M. Anderson et al., xe2x80x9cAdenovirus-mediated tissue-targeted expression of the HSVtk gene for the treatment of breast cancer,xe2x80x9d Gene Therapy, vol. 6, pp. 854-864 (1999). While these approaches are very promising, they require specific knowledge of the cancer cells, and are not applicable to most situations.
The use of suicide genes is one of the most promising strategies for gene therapy of solid tumors. Transfection of the herpes simplex virus type-1 thymidine kinase gene (HTK), given in combination with the drug ganciclovir (GCV), is the most commonly used cancer gene therapy system to date, both in experimental models and clinical trials. See J. Gomez-Navarro et al., xe2x80x9cGene therapy for cancer,xe2x80x9d European Journal of Cancer, vol. 35, pp. 867-885 (1999). HTK, whose substrate specificity is distinct from that of cellular thymidine kinases, can convert GCV to the toxic phosphorylated form, specifically killing the cells that express HTK. Since the concept of an HTK/GCV system was first described, it has shown good success as a tumor ablation strategy in a variety of experimental models. In addition, over two dozen clinical gene therapy trials based on this model have been initiated in the last seven years. See J. A. Roth et al., xe2x80x9cGene therapy for cancer: what have we done and where are we going?xe2x80x9d Journal of the National Cancer Institute, vol. 89(1), pp. 21-39 (1997); D. Klatzmann et al., xe2x80x9cA Phase I/II dose-escalation study of herpes simplex virus type 1 thymidine kinase xe2x80x9csuicidexe2x80x9d gene therapy for recurrent metastatic melanoma,xe2x80x9d Human Gene Therapy, vol. 9, pp. 2585-2894 (1998); and J. R. Herman et al.,xe2x80x9cIn situ gene therapy for adenocarcinoma of the prostate: A phase I clinical trial,xe2x80x9d Human Gene Therapy, vol. 10, pp. 1239-1249 (1999).
The HTK/GCV system is appealing due to its low inherent toxicity. Moreover, it has been shown that when as few as 10% of the cancer cells express HTK, it is still possible to obtain complete tumor ablation due to the xe2x80x9cbystander effectxe2x80x9d and specific immune responses. See R. Ramesh et al., xe2x80x9cIn vivo analysis of the xe2x80x98bystander effectxe2x80x99: a cytokine cascade,xe2x80x9d Experimental Hematology, vol. 24, pp. 829-838 (1996).
To direct the expression HTK primarily in cancer cells, most research to date has been based on either specific transcriptional regulation or specific delivery methods. Two examples of the former strategy are the use of tissue-specific promoters and inducible promoters. See Anderson et al., 1999; and Binley et al., 1999.
The protein eIF4E is the cap-binding subunit of the eIF4F complex, an ATP-dependent helicase that unwinds xe2x80x9cexcessxe2x80x9d secondary structure in the 5xe2x80x2 untranslated region (UTR) of mRNAs. The low-abundance of eIF4E/F is the limiting factor for the translation of some mRNAs, particularly those with long, G/C-rich 5xe2x80x2 UTRs with the potential to form a stable, secondary structure. See M. J. Clemens et al., xe2x80x9cTranslational control: the cancer connection,xe2x80x9d Int. J. Biochem. Cell Biol., vol. 31, pp. 1-23 (1999). Overexpression of eIF4E results in a specific increase in the translation of these weakly competitive mRNAs, many of which encode products that stimulate cell growth and angiogenesis, like FGF-2 and VEGF. See C. Kevil et al., xe2x80x9cTranslational enhancement of FGF-2 by eIF-4 factors, and alternate utilization of CUG and AUG codons for translation initiation,xe2x80x9d Oncogene, vol. 11, pp. 2339-2348 (1995); C. Kevil et al., xe2x80x9cTranslational regulation of Vascular Permeability Factor by eukaryotic initiation factor 4E: Implications for tumor angiogenesis,xe2x80x9d Int. J. Cancer, vol. 65, pp. 785-790 (1996); and P. A. E. Scott et al., xe2x80x9cDifferential expression of vascular endothelial growth factor mRNA versus protein isoforms expression in human breast cancer and relationship to eIF4E,xe2x80x9d British. J. Cancer, vol. 77, pp. 2120-2128 (1998).
Elevating eIF4E rescues the translation of repressed mRNAs with a complex 5xe2x80x2 UTR, many of which encode factors required for cell proliferation, e.g., protooncogene c-myc, cyclin D1, ornithine decarboxylase, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor (xe2x80x9cVEGF,xe2x80x9d otherwise known as vascular permeability factor, xe2x80x9cVPFxe2x80x9d). See A. De Benedetti et al., xe2x80x9ceIF4E expression in tumors: its possible role in progression of malignancies,xe2x80x9d Int. J. of Biochemistry and Cell Biology, vol. 31, pp. 59-72 (1999).
Overexpression of eIF4E has been shown to be ubiquitous in solid tumors, including bladder, breast, cervical, colon, head and neck, and prostate, as well as in many malignant cell lines. See J. P. Crew et al., Eukaryotic initiation factor-4E in superficial and muscle invasive bladder cancer and its correlation with vascular endothelial growth factor expression and tumour progression,xe2x80x9d Br. J. Cancer, vol. 82, pp. 161-166 (2000); V. V. Kerekatte et al., xe2x80x9cThe protooncogene/translation initiation factor eIF4E: a survey of its expression in breast carcinomas,xe2x80x9d Int. J. Cancer., vol. 64, pp. 27-31 (1995); I. B. Rosenwald et al., xe2x80x9cUpregulation of protein synthesis initiation factor eIF4E is an early event during colon carcinogenesis,xe2x80x9d Oncogene, vol. 18, pp. 2507-2517 (1999); C. O. Nathan et al., xe2x80x9cDetection of the proto-oncogene eIF4E in surgical margins may predict recurrence in head and neck cancer,xe2x80x9d Oncogene, vol. 15, pp. 579-584 (1997); Y. Miyagi et al., xe2x80x9cElevated levels of eukaryotic initiation factor eIF-4E mRNA in a broad spectrum of transformed cell lines,xe2x80x9d Cancer Letters, vol. 91, pp. 247-252 (1995); B. Anthony et al., xe2x80x9cOverexpression of the protooncogene- translation factor eIF-4E in breast carcinoma cell lines,xe2x80x9d Int. J. Cancer, vol.65, pp. 858-863 (1996); and I. B. Rosenwald, xe2x80x9cUpregulated expression of the genes encoding translation initiation factors eIF-4E and eIF-2alpha in transformed cells,xe2x80x9d Cancer Letters, vol. 102, pp. 113-23 (1996).
We have discovered a novel gene therapy for cancer, which unlike most prior appraches, does not require specific knowledge of the cancer cells, but instead targets a general characteristic that distinguishes cancer cells from normal cells, i.e., elevated eIF4E expression. The expression of a toxin or conditional toxin such as HTK is translationally repressed in normal cells by placing a complex 5xe2x80x2 UTR in front of its reading frame. In prototype experiments, this HTK mRNA, a transcriptional product of the BK-UTK vector, was translationally regulated so as to largely inhibit its production in normal murine and human cells, while cancer cells efficiently translated the protein, which a resulting increased sensitivity to GCV. Synthesis of the HTK protein from the BK-UTK vector (containing the 5xe2x80x2 UTR of fibroblast growth factor xe2x88x922 (xe2x80x9cFGF-2xe2x80x9d)) readily occurred in a panel of murine and human breast carcinoma lines, but not in normal cell lines. Subcutaneous tumors and experimental lung metastases of the breast carcinoma line MM2MT in BALB/c mice were greatly reduced by transfection with the BK-UTK vector, followed by GCV administration. Both the BK-UTK and the BK-TK (control) vectors were effective in reducing lung metastasis following systemic delivery of the vectors and subsequent GCV administration. However, the BK-TK vector was highly toxic to mice while little to no toxicity was seen in mice treated with the BK-UTK vector.